The purpose of this proposal is to explore the role of the TRPM2 ion channel in hepatic innate immunity, with a focus on hepatocytes. We have previously shown that TRPM2 is essential for mice to successfully overcome infections with Listeria monocytogenes (Lm). Based on the strong representation and known role of TRPM2 in cells of the myeloid lineage, we originally assumed that the absence of TRPM2 in immunocytes was the sole cause for high Lm susceptibility. However, the analysis of bone marrow chimeric animals revealed that the highly elevated listerial burdens in the liver, as opposed to the spleen, are the result of combined TRPM2-dependent functions both in the hematopoietic and parenchymal compartments. Strikingly, the early hepatocytic death that we found to be a hallmark of the TRPM2-/- phenotype of Lm-infected mice is solely dependent upon TRPM2's presence in the liver parenchyma. Lm is a facultative intracellular bacterial pathogen that target hepatocytes when injected intravenously, and mostly replicates in these cells. We thus hypothesize that TRPM2-/- hepatocytes are impaired in their ability to efficiently respond to Lm infection. To investigate this novel aspect of TRPM2 function, we propose to pursue following specific aims: 1) To define the role of TRPM2 in response to Listeria and related stimuli in hepatocytes, and 2) To define molecular determinants of TRPM2 function in hepatic innate immunity. Under the first aim, we will establish overexpression and inhibition of TRPM2 in a hepatocyte cell line. We will evaluate in this cell line, as well as in wildtype versus TRPM2-/- primary mouse hepatocytes, the impact of changing TRPM2 activity levels in response to Lm and pathogen recognition receptor agonists on various parameters such as cytokine production. Under aim 2, we will utilize TRPM2 mutants to perform a structure-function relationship study of TRPM2 in the context of hepatic anti-listerial defense, both in vitro and in vivo. We expect to gain insights about TRPM2 as a previously unsuspected signaling pathway in hepatic parenchymal and immune cells that could have relevance for many conditions leading to liver injury. The long-term goal of this research will be to manipulate TRPM2 function to influence hepatic innate immunity and inflammation, and treat or prevent clinical conditions such as liver fibrosis and cirrhosis.